DESCRIPTION (taken from the application) Early-onset type 2 diabetes (MODYs) are monogenic forms of NIDDM characterized by an early disease-onset and by defects in insulin secretion. Mutations in th genes encoding HNF4theta and HNF1theta lead to two clinically indistinguishabl forms of NIDDM (MODY1 and MODY3, respectively). HNF4theta is an upstream regulator of HNF1theta and both genes define a transcriptional hierarchy believed to be essential for normal pancreatic Beta-cell function. We have shown that HNF3beta is a strong transactivator of HNF4theta gene expression an that HNF3theta is a negative regulator of HNF4theta and downstream target gene transcription. This shows that HNF3theta and HNF3beta have antagonistic transcriptional regulatory functions in vivo and suggests that HNF3beta is the master regulator of a balanced transcription factor hierarchy essential for normal pancreatic islet function. We now propose to study the role of the HNF-transcriptional regulatory network in pancreatic islets in normal and diabetic conditions. We propose to generate transgenic and knockout mouse models in which the expression of HNF3theta and HNF3beta is altered. We will study gene expression and pancreatic islet function in these mouse models. Thi will include histological analysis, insulin secretion tests of perifused islet and perfused pancreata as well as gene expression studies of the HNF3/HNF4/HNF cascade and downstream targets by quantification of steady state mRNA and protein levels. In a complimentary approach, we propose to study the expressio of HNFs and downstream targets in islets of a rodent model of NIDDM, the Zucke diabetic fatty (ZDF) male rat, during the progression from the prediabetic to the diabetic state. Since we have previously shown that HNF4theta is a key regulator of expression of glycolytic enzymes and GLUT2 we also propose to tes if there is a correlation between expression of HNFs, their target genes and glucose responsiveness of pancreatic Beta-cell lines. Finally, we propose to use visceral endoderm which was derived from HNF3beta and HNF4theta null ES cells as a model system to identify novel HNF3 and HNF4theta target genes. Any novel targets will be characterized and expression will be studied in the mutant mouse models. We hope that a comprehensive analysis of the HNF-transcriptional hierarchy and its regulation of downstream target gene expression will lead to a better pathomolecular understanding of pancreatic islet function. Novel target genes of this pathway can in future be tested for genetic linkage or genetic variation in other forms of MODY/NIDDM and may be potential targets for therapeutic intervention aimed at preserving or improvin Beta-cell function.